Project Summary Tau is an intrinsically disordered and chemically heterogeneous protein whose misfolding into insoluble filamentous aggregates is one of the two hallmarks of Alzheimer's disease. A large body of biochemical evidence has shown that tau aggregates self-propagate, and suggest that different molecular conformations may underlie different fibril morphologies and neuropathological phenotypes of Alzheimer's disease. However, virtually nothing is known about the tau molecular structure and how it may vary in Alzheimer's disease to cause this disease diversity. We propose to use solid-state NMR spectroscopy to investigate the conformation and conformational variation of tau fibrils obtained in vitro and from seeding with Alzheimer's disease brain extracts. In Aim 1, we will map the structural variations of tau fibrils by measuring 13C and 15N NMR chemical shifts of amino-acid specifically labeled protein. Scattered labeled samples will probe whether a tau strain has a single conformation or multiple conformations, while consecutively labeled samples will probe the backbone conformation, ?-strand stacking, and protofilament packing at structural hotspots of the ?-sheet core. The NMR chemical shifts and cross peak patterns will be correlated with cell infectivity, proteolytic digestion pattern, fibril morphology and stability data to understand whether one strain has one unique conformation. In Aim 2, we will investigate how the fuzzy coat interacts with the repeat domain to shape the ?-sheet structure of the aggregate. 2D and 3D NMR experiments that correlate dynamic and rigid residues will be conducted. Water may act as the lubricant that mediates the fuzzy coat interaction with the ?-sheet core. We will conduct water-protein polarization transfer experiments to probe the water accessibilities on a residue-specific and domain-specific level. In Aim 3, we will determine the ?-sheet core structure of selected in vitro and brain-seeded tau samples. We will use 13C and 15N-detected 3D correlation NMR experiments under regular MAS rates and 1H-detected experiments under ultrafast MAS to obtain resonance assignment and interatomic distances. We will also develop 19F dipolar recoupling experiments to measure distances up to 15 to constrain the three-dimensional fold of the ?- sheet core. These studies will provide important residue-level insights into the molecular structural basis of tau toxicity in Alzheimer's disease, and should inform the development of therapeutic interventions to treat and prevent Alzheimer's disease.